The objective of the project is to study the role of heterotrimeric G proteins and their coupled receptors in normal cell growth and oncogenesis. We have genetically engineered NIH 3T3 mouse fibroblasts to express the family of human acetylcholine muscarinic receptors (mAChRs). Using this model, we have shown that genes for mAChRs subtypes coupled to the activation of phosphatidylinositol (PI) hydrolysis can act as ligand-dependent oncogenes, whereas those coupled to the inhibition of the adenylyl cyclase (AC) are not. We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein- coupled receptors. Our findings suggest that the Raf-1 kinase plays a critical role in transformation induced by this class of receptors, however these cell-surface receptors might also utilize signalling routes bypassing the requirement of Raf-1. We have observed that growth promoting pathways activated by receptors coupled to G proteins involve tyrosine phosphorylation of a small set of cellular proteins previously identified as substrates for oncogene-encoded tyrosine kinases, such as the p125FAK and p130 v-src substrates. We have studied the relationship between ras-GAP and G protein coupled receptors. Cotransfection of wild- type GAP prevented transformation by m1 mAChRs, whereas the mutant consisting of only its catalytic domain lacked any demonstrable effect. In contrast, the N-terminal non-catalytic domain of GAP effectively prevented m1-induced focus-formation. Thus, our findings suggest a role for the N-terminal non-catalytic domain of GAP in regulating biological functions mediated by G protein-coupled receptors. The recent discovery of a new family of G proteins, G12, distantly related to those that regulate the activity of adenylyl cyclases or PI-PLC prompted us to ask whether this novel class of G proteins harbors oncogenic potential. Our results show that, in contrast to alpha-i2 and alpha-q, overexpression of wild type alpha-12 in NIH 3T3 cells is itself weakly transforming, and mutationally activated alpha-12 behaves as one of the most potent oncogenes described to date. Transformation does not involve PI-PLC but correlates with alterations in other G protein-linked pathways. In addition, a systematic screen of tumor-derived cell lines suggests that alterations in the expression of this G protein might contribute to human neoplasia, particularly in adenocarcinoma of the breast and salivary glands.